Nisins

ABSTRACT

PCT No. PCT/GB83/00676 Sec. 371 Date Nov. 18, 1994 Sec. 102(e) Date Nov. 18, 1994 PCT Filed Apr. 1, 1993 PCT Pub. No. WO93/20213 PCT Pub. Date Oct. 14, 1993An organism which does not express its natural nisA gene product, but is capable of expressing genes for nisin modification, immunity and translocation out of the cell. The organism may be transformed with a coding sequence for a variant prenisin and appropriate regulatory sequences for expression thereof such that the organism is capable of secreting the corresponding variant nisin. A process for exclusively producing a variant nisin comprises fermenting this organism and obtaining the nisin so produced.

This application is a continuation, of application Ser. No 08/313,123,filed Nov. 18, 1994, now abandoned, which is a 371 application ofPCT/GB93/00676 filed Apr. 1, 1993.

This invention relates to the production of protein-engineered nisins.

Reference to FIG. 2 herein, refers to FIGS. 2A and 2B collectively.Reference to FIG. 6 herein, refers to FIGS. 6A and 6B collectively.Reference to FIG. 11 herein, refers to FIGS. 11A and 11B collectively.

Nisin is a highly modified peptide antibiotic produced, for example, bycertain strains of Lactococcus lactis. It is of great interest to thefood industry because of its efficient antimicrobial activity against awide range of gram-positive organisms including many spoilage bacteriaand food pathogens, for example, Listeria, Clostridia and Bacillusspecies (12,25).

The chemical structure of nisin is well established (6,17,34,45, FIG. 9)(SEQ ID NO:19). It is a member of the family of antibiotics termedlantibiotics. These unusual polycyclic peptides share the structuralfeatures of dehydro-residues and intrachain sulphide bridges forminglanthionine and β-methyllanthionine rings. The atypical residues areintroduced by post-translational modification of amino acids serine,threonine and cysteine in the primary sequence of a precursor peptide(lantibiotics are the subject of a recent extensive review, 26).Biosynthesis of nisin thus involves genes for both the inactiveprecursor of nisin, known as prenisin, (nisA) and also the modifyingenzymes responsible for nisin maturation. The mature nisin molecule isbased on a sequence of 34 amino acids (SEQ ID NO:19). The proteinencoded by nisA includes a 23 amino acid N terminal signal sequencewhich is cleaved off during secretion of nisin. The conversion ofprenisin, encoded by nisA, into mature nisin involves cleavage of theleader and the modification of individual amino acids. The nisA gene hasbeen cloned and characterised (1,27,11) and shown to have a chromosomallocation (11,42). A number of additional, as yet uncharacterised, genesinvolved in the enzymatic modification of prenisin, translocation andimmunity are encoded by nisin producing strains (42). Thesedeterminants, along with nisA, are thought to be clustered together ashas been described recently for the lantibiotics subtilin (28) andepidermin (41). It has been known for some time that nisin determinantscan be transferred by conjugation (14) and it has now been establishedthat this ability is due to their carriage on a large conjugativetransposon, Tn5301 (22,38).

Established protein engineering techniques can be used to introducechanges to the amino acid sequence of nisin. This involves modifying thecoding region of the nisin structural gene, nisA, for example bysite-directed or random mutagenesis. Expression of these changes iscomplicated by the fact that nisin is post-translationally modified.

Variant nisins may be constructed by the expression of variant nisAgenes in a host strain which encodes the necessary maturation machinery,and thus can process the modified precursor peptide. The simplestapproach is to transform a nisin producing strain with a recombinantplasmid encoding a variant nisA gene. In this background the host'smaturation enzymes are available to process both the resident prenisinand its plasmid-encoded variant. A strategy of this type has beenreported for a strain that carries the wild-type nisin transposon (29).However, the disadvantage of this system is that both the host's nisinand the engineered variant are synthesised together, making complexchemical separation procedures necessary prior to analysis of theproperties of the novel peptide. Such a procedure would be particularlyundesirable for industrial scale production of a variant nisin.

According to a first aspect of the present invention there is providedan organism which does not secrete its natural nisin, but is capable ofexpressing genes for nisin modification, immunity and translocation outof the cell.

By "an organism that does not secrete its natural nisin" we include anorganism which does not naturally encode a nisin. Examples of organismsthat do not naturally encode a nisin are Bacillus subtilis andEscherichia coli.

Preferably the organism is a lactococcal strain, most preferablyLactococcus lactis.

A second aspect of the present invention provides the organism describedabove transformed with a coding sequence for a variant prenisin and, ifnecessary, appropriate regulatory sequences for expression thereof, suchthat the organism is capable of secreting the corresponding variantnisin.

A third aspect of the present invention provides a process for producinga nisin comprising fermenting this organism and obtaining the nisinproduced thereby.

By "nisin" it is meant a peptide antibiotic produced by some naturallyoccurring nisin producing strains of bacteria. The mature molecule isbased on a sequence of amino acids encoded by a gene nisA.

By "variant nisin" it is meant a protein engineered variant of a naturalnisin in which changes to the amino acid sequence of the nisin have beenintroduced as a result of site-directed or random mutagenesis of thenisA gene.

Site-directed mutations of the nisA gene may be made, for example, bythe oligonucleotide-directed mutagenesis technique of Zoller & Smith(1983) Meth. Enzymol. 100, 468-500 and Zoller & Smith (1984) DNA 3,479-480 which uses mismatched oligonucleotide primers to introduce themutation.

It is convenient to use a method for improving the yield of mutants, forexample, the dut-ung method described by Kunkel (1985) Proc. Natl. Acad.Sci. USA 82, 488-492. Alternatively, the polymerase chain reaction (PCR)may be used to generate mutants using mismatched oligonucleotides (Saikiet al (1988) Science 239, 487-491).

Random mutants of the nisA gene can be made chemically using, forexample, sodium bisulphite or hydroxylamine as the mutagen.Alternatively, random mutations can be introduced into the nisA geneusing enzymatic misincorporation using a DNA polymerase with relativelylow fidelity, for example AMV reverse transcriptase or Taq DNApolymerase or by using mixtures of oligonucleotides, spiked duringsynthesis, to incorporate a small amount of each different bases at eachposition. These methods are well known in the art.

The derivation of an organism that expresses the maturation genes fornisin biosynthesis, but is deficient in its natural nisA gene product,thereby provides an effective means to produce variant nisins. Thetransformation of such a strain with a plasmid vector carrying anindividual nisA gene containing a site-directed or random mutationgenerates a strain that produces exclusively a variant nisin.

As the gene for nisA is only one of a coordinately expressed group ofgenes that also provide the nisin modification, immunity andtranslocation functions, simple inactivation of the nisA gene does notyield a strain that is able to convert the product of a plasmid-encodednisA gene into mature nisin.

A fourth aspect of the present invention provides a method ofconstructing an organism that does not secrete its natural nisin but iscapable of expressing the other nisin genes. The method comprisesselecting a nisin producing organism and selectively deleting the codingsequence for its natural nisA gene product or otherwise preventing thesecretion of the nisin polypeptide, for example, by modifying an aminoacid in the prenisin leader sequence, preferably amino acid 4.

A preferred method comprises insertionally inactivating the nisA geneand restoring the activity of the genes for nisin modification, immunityand translocation out of the cell. The restoration of activity may beachieved by selection in media containing nisin at inhibitory levels.

In another method the nisA gene is deleted or a mutation is introducedwhich prevents its translation and the expression of the associatednisin genes is maintained.

It will be appreciated by a person skilled in the art that an organismwhich does not naturally secrete nisin, and that does not naturallycontain genes for nisin modification, immunity and translocation out ofthe cell may be converted into an organism useful in the practice of thepresent invention by transferring into it the said genes for nisinmodification, immunity and translocation out of the cell. Thus, oneembodiment of the invention provides a means of converting an organismwhich is not useful in the practice of the invention to one that is.

For example, it is possible to transfer the nisin modification, immunityand translocation genes from the Lactococcus lactis strain FI 7332 (asdisclosed in the Example) to an organism that does not contain thesegenes, nor contains a nisA gene, for example E. coli or B. subtilis or asuitable lactococcal strain, by conjugation or transduction or by genecloning technology. It is preferred if the organism is B. subtilis or asuitable lactococcal strain.

Furthermore, it is possible to transfer the nisin modification, immunityand translocation genes from a strain not able to produce a nisin (forexample Lactococcus lactis strains FI 7300 and FI 7304 as disclosed inthe Example) to an organism that does not contain these genes, introducea nisA gene and then select for nisin production (that is, using similarmethods to those disclosed in the Examples for the production ofLactococcus lactis FI 7300). Such a transfer of genes may also be byconjugation or transduction or gene cloning technology (includingtransformation in the case of the nisA gene). It is preferred if theorganism is B. subtilis or a suitable lactococcal strain.

According to a fifth aspect of the present invention, the above-outlinedmethod further comprises transforming the organism with a codingsequence for a variant prenisin and, if necessary, appropriateregulatory sequences for expression thereof. The organism is capable ofpost-translational modification of said prenisin and secretion of thecorresponding variant nisin.

A sixth aspect of the present invention provides a nisin having aresidue other than dehydroalanine corresponding to the residue in thefirst ring of natural nisin derived from the serine residue ofunmodified prenisin. Said first ring of mature nisin is shown as ring ain FIG. 9 (SEQ ID NO:15).

A seventh aspect provides a nisin which does not have a dehydroalanineresidue in the first ring. This variant nisin is preferably producedusing the expression system of the present invention. A study of nisinbreakdown during storage has revealed that activity is lost when thefirst ring of mature nisin becomes opened at amino acid 5 (5,34). Thisresidue is dehydroalanine in mature nisin and it is derived bydehydration of a serine residue in prenisin. It is believed that theopening of the ring is due to the fact that the dehydroalanine islabile. A site-directed mutant of the nisA gene may be constructed inwhich the codon for serine at residue 5of the natural prenisin ischanged to one that encodes an amino acid other than serine. Forexample, codon for serine 5 may be changed to encode alanine, valine,threonine, leucine, isoleucine, glycine, histidine, arginine, lysine,aspartate, glutamate, asparagine, glutamine, proline, methionine,cysteine, phenylalanine, tyrosine or tryptophan. Alanine is preferred.The expression of the engineered nisA gene in a nisin producing organismin accordance with the present invention leads to the production of avariant mature nisin. In the case of the preferred mutation to analanine codon, the variant mature nisin is named nisinA S5A.Investigations indicate that this variant nisin has greater stabilitythan natural nisin A and is therefore an example of the exclusiveproduction of an improved nisin.

The variant nisin not having dehydroalanine at position 5 may have othervariations as well, although it is preferable to retain thedehydroalanine residue at position 33.

The amino acids replacing serine at position 5 may be modifiedpost-translationally to form an amino acid not coded for directly, forexample as disclosed herein for threonine residues.

Evidently, it is also possible to produce this variant nisin byexpressing the engineered variant nisA gene on a plasmid vector that istransformed into a normal nisin producing organism. Mature variant nisinis produced as well as normal nisin.

It may also be possible to chemically synthesise the variant nisinpeptides wherein Serine 5 is replaced by another amino acid residue.

Peptides, such as the variant nisins, may be synthesised by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Luet al (1981) J. Org. Chem. 46, 3433 and references therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is effected using 20% piperidine in N,N-dimethylformamide.Side-chain functionalities may be protected as their butyl ethers (inthe case of serine threonine and tyrosine), butyl esters (in the case ofglutamic acid and aspartic acid), butyloxycarbonyl derivative (in thecase of lysine and histidine), trityl derivative (in the case ofcysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (inthe case of arginine). Where glutamine or asparagine are C-terminalresidues, use is made of the 4,4'-dimethoxybenzhydryl group forprotection of the side chain amido functionalities. The solid-phasesupport is based on a polydimethylacrylamide polymer constituted fromthe three monomers dimethylacrylamide (backbone-monomer),bisacryloylethylene diamine (cross linker) and acryloylsarcosine methylester (functionalising agent). The peptide-to-resin cleavable linkedagent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acidderivative. All amino acid derivatives are added as their preformedsymmetrical anhydride derivatives with the exception of asparagine andglutamine, which are added using a reversedN,N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used are ethanedithiol, phenol, anisole and water,the exact choice depending on the constituent amino acids of the peptidebeing synthesised. Trifluoroacetic acid is removed by evaporation invacuo, with subsequent trituration with diethyl ether affording thecrude peptide. Any scavengers present are removed by a simple extractionprocedure which on lyophilisation of the aqueous phase affords the crudepeptide free of scavengers. Reagents for peptide synthesis are generallyavailable from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.Using this chemical synthetic method it is possible to introducenon-natural amino acids into the synthetic nisin variant. Purificationmay be effected by any one, or a combination of, techniques such as sizeexclusion chromatography, ion-exchange chromatography and (principally)reverse-phase high performance liquid chromatography. Analysis ofpeptides may be carried out using thin layer chromatography,reverse-phase high performance liquid chromatography, amino-acidanalysis after acid hydrolysis and by fast atom bombardment (FAB) massspectrometric analysis.

Alternatively, nisin variants, including those with an "unnatural" aminoacid replacing serine at position 5, may be produced by in vitrocell-free translation.

Other variant nisins may also be exclusively produced using theexpression system of the present invention. Retaining a dehydroalanineresidue at positions 5 or 33 is preferred.

An active natural variant of nisin, known as nisin Z, has asparagineinstead of histidine at amino acid 27 (15,37). A variant of nisin Z, forexample, in which the amino acid corresponding to asparagine is changedto glutamine may be produced using a site-directed mutant of the nisAgene in the expression system. This variant nisin, named nisinA H27Q,has full biological activity.

Further aspects of the present invention provide the variant nisinsproduced using the expression system of the present invention, and theuse of such nisins as antimicrobial agents.

The variant nisins of the present invention are particularly useful asantimicrobial agents in those conditions where the natural nisin isring-opened and loses its antimicrobial properties. Such conditionsinclude those of high pH present in many food situations and where theactivity of normal nisin is limited. Preferred organisms to be killedinclude Listeria monocytogenes and Gram negative bacteria.

So that the invention may be more readily understood, preferred aspectswill now be illustrated by way of example, and with reference to theaccompanying drawings in which:

FIG. 1 illustrates the strategy for nisA gene replacement.

It shows a) a map of the gene replacement vector pFI283 and maps showingequivalent regions of chromosome encoding nisA and flanking sequences instrains b) FI5876, c) FI7181 and d) FI7300. The thin line representsplasmid DNA and the thick line represents lactococcal chromosomal DNA.The nisA and nisB genes are indicated by black boxed regions and DNAsequences containing the erythromycin resistance (Em^(r)) determinantare shown as shaded boxes. The direction of transcription of the genesis indicated by arrows above the maps. The small numbered arrows belowthe maps represent primers used in PCR analysis. A single recombinationevent between lactococcal sequences to the left of the Em^(r)determinant on pFI283 and homologous sequences on FI5876 (X) results inCampbell integration of the plasmid with the organisation of sequencesas shown in FI7181. Recombination between pFI283 sequences on both sidesof the Em^(r) determinant and homologous FI5876 sequences (X and Y)leads to gene replacement, as found with FI7300.

FIG. 2 shows the double stranded nucleotide sequence of the nisA geneand flanking regions (SEQ ID NO:1)

Coding regions preceded by ribosome binding sites (RBS) are indicatedand the primary translation products (SEQ ID NO:2) are given below thesequence. The arrow between the arginine (R) at codon 154-156 andisoleucine (I) at codon 157-159 shows the point of cleavage of theN-terminal leader sequence of prenisin. Shaded regions represent thenucleotide sequences of synthetic oligomers employed as primers forPCR-mediated site-directed mutagenesis. The 3' end of the primers andthe direction in which PCR proceeds is indicated by numbered arrows. Thenumbers refer to those primers as listed in methods. Specific mismatchesincluded in the primers are represented by shaded nucleotides above orbelow the sequence. Primers 7 and 8 (SEQ ID NOS 14and 15, respectively)are designed to substitute a glutamine (Q) codon for a histidine (H)codon (coordinates 235-237) Primer 11 (SEQ ID NO:18) contains an alanine(A) codon in place of a serine (S) codon (coordinates 169-171). Theprimers containing restriction sites at their 5' ends define the terminiof either PCR generated SacI/EcoRI restriction fragments (primers 5 and6) (SEQ ID NOS 12 and 13, respectively) or BamHI/SacI restrictionfragments (primers 9 and 10 or 9 and 11) (SEQ ID NOS 16 and 17, or SEQID NOS 16 and 18, respectively). The base changes mentioned above areincorporated in these fragments, which are then cloned into theexpression vectors (see FIGS. 3 and 4) to reconstitute nisA genescontaining the specific mutation.

FIG. 3 shows the construction of nisA expression vectors which use thenatural nisin gene promoter.

The linear plasmid maps are of a) pFI172, b) pFI354, c) pFI378 and e)pFI411. Cloned Lactococcal DNA is represented by a thick line and vectorsequences are indicated by a thin line. Genes within the clonedsequences are boxed and relevant restriction sites are given above themaps. d) illustrates the strategy employed for site-directed mutagenesisof the nisA gene to introduce the amino acid substitution His²⁷. Doublestranded DNA sequence between the SacI and EcoRI sites shown in pFI378(c) is represented by a double line. The sites at which primers annealare indicated by arrows above and below the lines and the fragmentswhich are amplified by PCR using combinations of these primers arerepresented by single thin lines. The mismatches incorporated in primers7 and 8 (SEQ ID NOS 14 and 15, respectively) (see FIG. 2), and themutation they generate in pFI411 (e), are indicated with an asterisk.

FIG. 4 shows the construction of nisA expression vectors forsite-directed mutagenesis throughout the nisA gene using the lacpromoter.

A PCR generated BamHI fragment, encoding the lacr gene and thedivergently transcribed lacR and lacA promoters (18, ref), was digestedwith XmnI. The 0.45 kb restriction fragment (see FIG. 5) was subclonedinto pTG262 to generate pFI451(a). Fragments N and C containing the N-and C-terminal regions of the nisA gene respectively, were constructedby PCR and cloned into pTG262 to generate pFI446(b). The nisA cassettecontains the uninterrupted nisA gene preceded by 60 bp, including theRBS, and followed by an intergenic gap and the start of the nisB gene(SEQ ID NO:1as shown in FIG. 2). Vector pFI449 (c) contains the nisAcassette cloned into pFI451 as a BamHI/EcoRI restriction fragment.Vector pFI480 (d) contains the N-fragment of the nisA cassette clonedinto pFI451 as a BamHI/SacI fragment. Vector pFI487 (e) contains theC-fragment of the nisA cassette cloned into pFI451 as a SacI/EcoRIfragment.

FIG. 5 shows the nucleotide sequence of the lactose operon promoter (SEQID NO:33)

The XmnI/BamHI fragment contains the lacA promoter and start of the nisAgene in the nisA cassette.

FIG. 6 shows plate diffusion bioassays.

Agar is seeded with the indicator strain Lactobacillus helveticus CH-1and the wells contain samples of supernatants from various Lactococcuslactis subsp. lactis strains. Plate 1 shows that nisin A is produced bythe FI7332 expression system as a result of complementation of the hostsnisA deficiency by plasmid encoded nisA genes. Furthermore, biologicalactivity of the variant nisin A/H27Q is demonstrated. Wells containsupernatants from FI7330 (FI7332/pFI172 plasmid encoded nisA); FI5876(nisin producing parent strain); MG1614 (non-nisin producing strain);FI7332/pFI411 (plasmid encoded nisA/H27Q); FI7332/pFI378 (plasmidencoded nisA); FI7332 plasmid free; and FI7332/pFI354 (plasmid vectorfor construction of variant nisA genes). Plate 2 demonstrates nisinactivity by strains encoding nisA and the variant nisA/S5A gene underthe control of the lacA promoter. Wells contain supernatants from FI5876(nisin producing parent strain); FI7332 (plasmid free); FI7332/pFI378(plasmid encoded nisA, natural promoter); FI7332/pFI449 (plasmid encodednisA, lacA promoter); and FI7332/pFI493 (plasmid encoded nisA/S5A, lacApromoter). 100 μl of the following nisin standards (dissolved in 0.02MHCL) were loaded into some wells: 500 U/ml; 300 U/ml; 200 U/ml; 100U/ml; and 0 U/ml.

FIG. 7 shows the construction of a host strain for the exclusiveexpression of mutant nisA genes.

Maps are of equivalent chromosomal regions of a) the nisin producingstrain FI5876 and derivatives b) FI7300, c) FI7304 and d) FI7332. ThenisA gene and the start of nisB gene are indicated by black boxes. Theinsertions in nisA are signified by a shaded box (Em^(r), erythromycinresistance) and an open box (IS905). Primers employed for analysis ofnisA insertional inactivation are shown as numbered arrows below themaps. Lines connecting primers represent the amplified fragment withsizes given in kilobases.

FIG. 8 shows PCR analysis of the constructed host strains.

PCR fragments generated with primers 1 and 2 (SEQ ID NOS 8 and 9,respectively) are separated by agarose gel electrophoresis. Template DNAfrom the non-nisin producing strain MG1614 is not amplified (track 2)whereas a band of 0.9 kb is generated from the nisin producing parentstrain FI5876 (track 3). This band is also present when DNA from FI7181is employed as template. However, Campbell integration of the genereplacement vector pFI283 in this strain (see FIG. 1c) results in thegeneration of a second band of 1.9 kb (track 4). In FI7330 (track 5) thenisA gene has been insertionally inactivated as a result of genereplacement (see FIG. 1d) and the 0.9 kb band is replaced with the 1.9kb band containing the integrated Em^(r) marker. This band is increasedin size by a further 1.3 kb to 3.2 kb in FI7304 (track 6) as a result ofinsertion of IS905 in the Em^(r) gene (see FIG. 7c). Strain FI7332 hasundergone a 200 bp deletion at one end of IS905 (FIG. 7d) and acorresponding decrease in the equivalent PCR generated fragment resultsin a 3.0 kb band (track 7). Tracks 1 shows λDNA digested with BglIincluded as a size standard. The 1.2% agarose gel was run for 2.5 hoursat 100 volts.

FIG. 9 shows the structure of nisin A (SEQ ID NO:19).

The modified residues are dehydroalanine (Dha), dehydrobutyrine (Dhb),aminobutyrate (Abu), lanthionine (Ala-S-Ala) and β-methyllanthionine(Abu-S-Ala). The predicted molecular alterations in Nisin A/H27Q andNisin A/S5A are shown as amino-acid substitutions in ring e and ring arespectively.

FIG. 10 illustrates the presence of multiple copies of DNA in thelactococcal genome.

Southern transfer hybridizations of restriction enzyme digestedchromosomal DNA from MG1614 (tracks 1 & 3) and FI5876 (tracks 2 & 4).Restriction endonucleases HincII (tracks 1 & 2) and PvuII (tracks 3 & 4)were used. The filter was probed with a ³² P-labelled gel-purified PCRfragment generated with primers 3 and 4 (SEQ ID NOS 10 and 11,respectively) using FI7304 DNA as template (see FIG. 7c). Multiple bandsindicate that there are several regions that display homology to theprobe which is characteristic of multicopy IS elements in the chromosomeof these Lactococcal strains.

FIG. 11 shows the single stranded nucleotide sequence of insertionsequence IS905 (SEQ ID NO: 4) and its encoded polypeptide sequence (SEQID NO: 5).

The inverted repeats which define the termini of the element areunderlined.

FIG. 12 shows the potential promoter active sequences at junctionsgenerated by IS905 insertion in strains a) FI 7304 (SEQ ID NOS:6 and 20,respectively) and b) FI 7332 (SEQ ID NOS 7 and 21, respectively)

EXAMPLE

Microbiological techniques and strains used: Most Lactococcus lactissubsp. lactis strains, generated in the course of this work, werederived from the nisin producing strain L. lactis FI5876 (11,22). Theconstruction of the derivative strains and their relevant properties aredescribed in the results and in Table 1. The plasmid-free, non-nisinproducing strain MG1614 (13) was included as a control. L. lactisstrains were routinely grown at 30° C. in M17 media (43) supplementedwith 0.5% (w/v) glucose (GM17). Selection for antibiotic resistancemarkers was as follows:- chloramphenicol resistance (Cm^(r)), 5 μg/ml;erythromycin resistance (Em^(r)) was induced at the subinhibitory levelof 50 ng/ml followed by selection at 5 μg/ml.

                  TABLE 1                                                         ______________________________________                                        Characterisation of nisin producing strain FI5876 and derivatives.                                           Nisin                                                               Nisin     Immunity.sup.b                                 Strain    Em.sup.r   Production.sup.a                                                                        (IU/ml)                                        ______________________________________                                        MG1614    -          -         <10                                            FI5876    -          +         1-3 × 10.sup.3                           FI7300    +          -         2-5 × 10.sup.2                           FI7304    -          -         1-3 × 10.sup.3                           FI7332    -          -         1-3 × 10.sup.3                           ______________________________________                                         .sup.a nisin production was determined by the plate diffusion assay.          .sup.b levels at which strains were immune to nisin are given between two     values. At the lower level growth was unaffected but inhibition of growth     was evident at the upper level.                                          

The Escherichia coli strain MC1022 (3) was the host strain used forconstruction of recombinant plasmids. Cultures were propagated at 37° C.in L broth (30). Selection for ampicillin resistance (Ap^(r)) was at 100μg/ml and Cm^(r) was at 15 μ/ml.

Determination of nisin production by L. lactis strains was based on theplate diffusion assay of Tramer and Fowler (44). Lactobacillushelveticus CH-1(Chr. Hansens Labs, Danmark A/S) was used as the nisinsensitive indicator strain. 0.5 ml of an overnight culture, grown in MRSmedia (9), was used to seed 50 ml MRS agar (pH 6.0) containing 1 mlTween-20/Ringers solution (50:50). Wells were loaded with 100 μl of testsample and the plates incubated at 4° C. for a minimum of 3 hours (toallow diffusion) prior to overnight incubation at 42° C.

Nisin immunity was determined by streaking a loopful of stationary-phasecells on plates containing varying amounts of nisin. Nisin (Koch-Light),dissolved in 0.2 M HCl, was added to GM17 agar up to a maximumconcentration of 5×10³ U/ml at which growth of all strains wasinhibited. L. lactis strains were considered to be immune to the highestlevel of nisin at which growth was evident throughout the streak.

Plasmid curing was achieved by growth in the presence of acriflavine(36).

Transformation: Recombinant plasmids were recovered by transformation ofE. coli by the method of Cohen et al, 1972 (8) with the modification ofHumphreys et al, 1979 (24). L. lactis strains were transformed byelectroporation as described by Holo and Nes, 1989 (19) with thefollowing modifications. Cells were grown in GM17 broth supplementedwith 2% glycine and selection was made on GM17 plates containingantibiotic. Sucrose was omitted from the initial growth media and theselection plates. Electroporation was performed using the Gene Pulserapparatus (Bio-Rad).

Molecular techniques: Total genomic DNA from L. lactis strains wasprepared according to the method of Lewington et al, 1987 (33). PlasmidDNA was isolated by the SDS alkaline lysis method. Purification of CCCDNA was by CsCl/EtBr gradient centrifugation (35). Restriction enzymesand other DNA modifying enzymes from various sources were used accordingto the suppliers recommendations. Conditions employed for PCR analysiswere as described previously (22). The following primers were used inthis study:

    ______________________________________                                        1.  5'-AAGAATCTCTCATGAGT; (SEQ ID NO:8)                                       2.  5'-CCATGTCTGAACTAACA; (SEQ ID NO:9)                                       3.  5'-GTGGAATACGGGTTTG; (SEQ ID NO:10)                                       4.  5'-TAAATAATTTATAGCTATTG; (SEQ ID NO:11)                                   5.  5'-CAGAGCTCTGATGGGTTG (SacI site underlined);                                 (SEQ ID NO:12)                                                            6.  5'-GTAGAATTCCGTTTATCGTTTGGAG (EcoRI site                                      underlined); (SEQ ID NO:13)                                               7.  5'-GCAACTTGTCAGTGTAGTATTCAC; (SEQ ID NO:14)                               8.  5'-GTGAATACTACACTGACAAGTTGC; (SEQ ID NO:15)                               9.  5'-AACGGATCCGATTAAATTCTGAAGTTTG (BamHI site                                   underlined); (SEQ ID NO:16)                                               10. 5'-TCAGAGCTCCTGTTTTACAA (SacI site underlined);                               (SEQ ID NO:17)                                                            11. 5'-TCAGAGCTCCTGTTTTACAACCGGGTGTACATAGTGCAAT                                   (SacI site underlined); (SEQ ID NO:18)                                    ______________________________________                                    

All PCR amplified fragments generated were initially cloned into pUC18(47) and the nucleotide sequence confirmed prior to vector construction.Nucleotide sequence determination of plasmid DNA was performed by thedideoxy chain termination method (40). Sequenase Version 2.0 was usedaccording to the suppliers recommendations (United States BiochemicalCorp.).

Site-directed mutagenesis was carried out using either PCR-mediatedoverlap extension or by incorporation of a specific mismatch into theparticular primer used in the nisA fragment amplification.

Southern blot hybridisation and labelling of probes was performedaccording to published techniques (22).

Results

Insertional inactivation of the chromosomally located nisA gene: Thegene replacement vector pFI283 was constructed to insertionallyinactivate the chromosomally encoded nisA gene. It carries a cloned nisAgene which is disrupted by the insertion of an erythromycin resistancegene (FIG. 1a). Plasmid construction involved the following steps:

The nisA gene of FI5876 was cloned into the shuttle vector pTG262 togenerate pFI172 which has been described previously (11,22). A 2 kbAccI/SalI fragment from this construct, containing nisA and the start ofnisB, was subcloned into the pBR322 based vector, pMTL23P (4,46). A 1 kbfragment encoding the erythromycin resistance gene of the staphylococcalplasmid pE194 (20) was inserted into the unique SacI site within thecloned nisA gene. This insertion resulted in disruption of the nisAgene. The erythromycin resistance gene in this construct was transcribedin the same direction as the nisA gene and was flanked on either side byapproximately 1 kb of lactococcal DNA sequences. A unique EcoRV site inthe adjacent polylinker of the vector sequences was used to insert a 2kb fragment carrying the chloramphenicol resistance gene originatingfrom the staphylococcal plasmid pC194 (21). A map of the resultingrecombinant plasmid, pFI283, is shown in FIG. 1a.

The nisin producing strain L. lactis FI5876 was transformed with pFI283and erythromycin resistant transformants were obtained. The plasmid doesnot encode a replication origin functional in Lactococcus and hence therecovery of erythromycin resistance in these transformants requiredintegration of this marker into the recipient chromosome.

Reciprocal recombination between homologous sequences on pFI283 (FIG.1a) and the chromosome of FI5876 (FIG. 1b) could generate two types oftransformants. A single cross-over event would result in the entireplasmid integrating in the chromosome (Campbell integration). A doublecross-over event, one on either side of the erythromycin resistance genein pFI283, would exchange the wild-type chromosomal nisA gene for theinsertionally inactivated copy, with subsequent loss of chloramphenicolresistance encoded by the non-replicating plasmid (gene replacement).Both recombination mechanisms have been shown to operate in Lactococcuslactis (7,31,32). The two alternative types of recombination could bedistinguished phenotypically in the transformants obtained by screeningfor chloramphenicol resistance. Transformants with only erythromycinresistance were recovered in which gene replacement had taken place(FIG. 1d). Strain FI7300 is typical of this construct.

The chromosomal rearrangement was confirmed by PCR analysis usingprimers 1 and 2 (SEQ ID NOS 8 and 9, respectively) which specificallyamplify a 0.9 kb fragment from FI5876 chromosomal sequences containingthe nisA gene and flanking regions (FIG. 1b; FIG. 8, track 3). When DNAfrom FI7300, was used as template a 1.9 kb fragment was amplified byprimers 1 and 2 (SEQ ID NOS 8 and 9, respectively) (FIG. 8, track 5). A1 kb increase in size of this fragment would be expected if theerythromycin resistance gene was integrated in this part of thechromosome (FIG. 1d) and is consistent with the proposal that genereplacement had substituted the parental wild-type nisA gene with theinsertionally inactivated copy.

Strain FI7300 having lost the parental nisA gene as a consequence ofgene replacement no longer produced nisin. Furthermore the insertion inthe nisA gene in this strain affected the nisin immunity level which wasreduced to below 500 U/ml (Table 1).

In an attempt to recover nisin production in the nisA deficient hostFI7300 the strain was transformed with pFI172, which encodes nisA, andsix transformants were tested for nisin production. The bioassays gavenegative results indicating that the chromosomal mutation in this hostcould not be complemented by provision of the nisA gene product in trans(Table 2).

Activation of genes for nisin immunity and modification: Reduction innisin immunity due to the insertional inactivation of nisA in FI7300 maybe caused by a polar effect on downstream genes. This may also result inreduced expression of genes required for modification thus preventingcomplementation of the nisA mutation in this host. In order to selectderivatives of FI7300 in which the immunity and maturation genes werefully active mutation to wild-type levels of nisin immunity was selectedby growth in media containing nisin at inhibitory levels. Colonies whichgrew on agar plates containing 10³ μg/ml nisin were picked and the cellsgrown on in media containing the same level of nisin. One such mutant,designated FI7304, expressed wild-type levels of nisin immunity, did notproduce nisin and furthermore was no longer resistant to erythromycin(Table 1).

PCR analysis of FI7304 DNA, using primers 1 and 2 (SEQ ID NOS 8 and 9,respectively) resulted in amplification of a 3.2 kb fragment (FIG. 8,track 6), 1.3 kb larger than the equivalent FI7300 fragment generated bythe same primers (FIG. 8, track 5). This suggested that loss oferythromycin resistance was not caused by a deletion in this region ofthe genome. The retention of the erythromycin resistance gene sequenceswas confirmed by PCR using primers 3 and 4(SEQ ID NOS 10 and 11,respectively), which are specific for a region at the 3' end of thatgene and amplify a 0.4 kb fragment (FIG. 7b). As these primers generatedan FI7304 fragment 1.7 kb in size it was concluded that an additional1.3 kb of DNA was inserted in this region of the erythromycin resistancegene (FIG. 7c). This insert results in loss of erythromycin resistancewith concurrent recovery of nisin immunity (see below). A comparison offragments from FI7300 and FI7304 generated by amplification betweenprimers 3 and 2 (SEQ ID NOS 10 and 9, respectively) and primers 3 and 4(SEQ ID NOS 10 and 11, respectively) was consistent with thisinterpretation (FIG. 7b and c).

The extra DNA sequences gained by FI7304 were amplified with primers 3and 4 (SEQ ID NOS 10 and 11, respectively) (FIG. 7c) and this PCRfragment was used to probe a southern blot of restriction enzymedigested genomic DNA from the parent strain, FI5876. A number offragments hybridised to the probe (FIG. 10) indicating that theadditional DNA in FI7304 is present in multiple copies in the genome ofthis strain. Further investigation has revealed that these repeatedsequences represent a new lactococcal insertion sequence designatedIS905, the sequence of which is presented in FIG. 11. As has beendemonstrated for other IS elements (2,39,48), transcriptionalread-through from a potential promoter within IS905 may lead to turn-onof downstream genes. The IS element IS905 was found to have significanthomology with IS256, which is known to express an adjacent antibioticresistance gene from an internal promoter (2,39). Such promoter activitycould account for the observed increase in nisin immunity exhibited byFI7304 which is equivalent to that of the parent strain, FI5876(Table 1) and it may also have restored the expression of genes requiredfor processing of prenisin to a level sufficient to facilitate nisAcomplementation. The DNA sequences of the junction of IS905 and the Emgene in FI 7304 and of IS905 and the nisA gene in FI 7332 are shown inFIG. 12.

Expression and maturation of plasmid encoded nisA: FI7304 wastransformed with the nisA encoding plasmid, pFI172. Transformants wereobtained at low frequency and the majority did not produce nisin inbioassays. One transformant, designated FI7330, was found to yield nisinat a level approximately 50% that of the parent strain FI5876 (FIG. 6).It was presumed that FI7330 had undergone a spontaneous mutation, eitherwithin the plasmid or the chromosome sequences, which resulted in nisinproduction. Isolation of plasmid DNA from FI7330 yielded a moleculeindistinguishable from pFI172 on the basis of restriction enzymeanalysis. Curing FI7330 of plasmid DNA, to generate the plasmid freestrain FI7332, resulted in loss of nisin production (FIG. 6). However,when plasmid pFI172 was introduced back into the latter strain hightransformation frequencies were obtained (FIG. 12) and all transformantsproduced nisin in bioassays.

When DNA from the plasmid free strain, FI7332, was analysed by PCR usingprimers 1 and 2 (SEQ ID NOS 8 and 9 respectively) a small size reduction(200 bp) was observed in the amplified fragment. The deletion was in thevicinity of the IS905 insertion in FI7304 (FIG. 8, cf tracks 5 and 6).Primers 3 and 4 (SEQ ID NOS 10 and 11 respectively) (derived from theerythromycin resistance gene sequences, (FIG. 7b) did not generate afragment with this template (FIG. 7d) indicating that the deletedsequences in FI7332 included a region at the 3' end of the erythromycinresistance gene into which IS905 had inserted. The deletion does notextend beyond the nisA gene as primer 2 (SEQ ID NO:9) (specific forsequences at the end of nisA), together with either primers 1 or 3 (SEQID NOS 8 and 10 respectively), resulted in fragment amplification (FIG.7d). The small chromosomal rearrangement in FI7332 has not affectednisin immunity which is conferred at a level indistinguishable from thatof the parent strain (Table 1).

Expression of variant nisA genes: To produce engineered nisin moleculesPCR-mediated site-directed mutagenesis was carried out on the nucleotidesequence of the nisA gene. The expression vectors pFI354 (FIG. 3b) andpFI451 (FIG. 4a) were designed for the expression of site-directedmutants of the nisA gene.

Plasmid pFI354 was constructed by deletion of a 1.25 kb SacI fragmentfrom pFI172. It thus encodes only the N-terminal part of nisA (FIG. 3b).Using PCR the C-terminal part of the nisA gene was amplified as a 254 bpSacI/EcoRI fragment. By cloning this fragment into pFI354 an intact nisAgene could be reconstituted as in pFI378 (FIG. 3c). Adaptation of thisprocedure enabled predetermined substitutions to be introduced into theC-terminal region of the nisA gene (using site-directed mutagenesis, seebelow). These pFI354 derivatives utilize the natural promoter of thenisin operon which lies between the SalI site defining one end of thecloned chromosomal fragment and the start of the nisA gene (FIG. 3). Inthis system the incorporation of site-directed mutations is limited tosequences in the nisA coding region downstream of the SacI site (FIG. 2)(SEQ ID NOS 1 and 2, respectively).

Initially the nisA codon selected for alteration was Histidine²⁷ whichlies within ring 5 of the mature nisin A molecule (FIG. 9) (SEQ IDNO:19). A naturally occurring variant of nisin A, termed nisin Z, hasbeen identified which contains an Asparagine residue in place ofHistidine²⁷ (15,23,37). The engineered incorporation of a similarlycharged Glutamine in place of Asparagine at residue 27 would thusrepresent a conservative substitution in the nisin Z amino acidsequence. The mutation involved a single base pair change in the PCRgenerated SacI/EcoRI fragment carrying the C-terminal end of nisA (FIG.2) (SEQ ID NOS 1 and 2, respectively). The reconstituted gene,containing a Glutamine codon (CAG) in place of a Histidine codon (CAT.FIG. 2) (SEQ ID NOS 1 and 2, respectively) was designated nisA/H27Q inaccordance with agreed nomenclature (10). Site-directed mutagenesis wascarried out using PCR-mediated overlap extension (FIG. 3d) as follows:

Terminal primers 5 and 6 (SEQ ID NOS 12 and 13, respectively) contain aSacI site and an EcoRI site respectively and define the ends of a 254bpfragment encoding the C-terminal 20 amino acids of prenisin (FIG. 2)(SEQ ID NOS 1 and 2, respectively). A pair of overlapping complementaryprimers (7 and 8) (SEQ ID NOS 14 and 15, respectively) were designedfrom sequences within the C-terminal region of nisA which included asingle base change from the original nisA sequence (FIG. 2) (SEQ ID NOS1 and 2, respectively). These were used in PCR amplifications inconjunction with one of the terminal primers (FIG. 3d) to create twopartially complementary fragments with the specific mutation located inthe overlapping region. The fragments were annealed to provide atemplate for subsequent PCR involving the same terminal primers thatdetermined the two ends of the SacI/EcoRI fragment (primers 5 and 6,(SEQ ID NOS 12 and 13, respectively) FIG. 3d). The final PCR generatedfragment containing the specific mutation was purified by isolation froman agarose gel using DEAE-NA 45 membrane (Schleicher and Schuell).Modification of ragged ends was carried out using T4 DNA polymerase andpolynucleotide kinase. The blunt ended fragment was cloned into the SmaIsite of pUC18 and the nucleotide sequence of the manipulated regiondetermined to confirm that the selected mutation was present. ASacI/EcoRI fragment from the pUC18 derivatives was then subcloned intopFI354 to recover an uninterrupted nisA reading frame containing thepredetermined mutation. This plasmid was designated pFI411 (FIG. 3e).

Vectors pFI480 and pFI487 were constructed in which expression of thenisA gene was under the control of the lacA promoter (FIG. 4). In orderto introduce changes throughout the entire nisin molecule a nisA genecassette was constructed. Using PCR with primers 9 and 10 (SEQ ID NOS 16and 17, respectively) (FIG. 2) the N-terminal part of the nisA gene wasamplified as a 200 bp BamHI/SacI fragment. This fragment was cloned intoplasmid pFI354 upstream of the 254bp SacI/EcoRI fragment encoding theC-terminal part of nisA. The latter was generated by PCR using primers 5and 6 (SEQ ID NOS 12 and 13, respectively) as described above. In thisway an intact coding region for the whole nisA gene, lying within a 448bp BamHI/EcoRI fragment, was reconstituted from two PCR generatedfragments which encoded the N and C parts of the nisin molecule. Theresultant plasmid was designated pFI446 (FIG. 4). The nisA cassetteincludes the upstream nisA ribosome binding site and the start of thenisB gene (FIG. 2) (SEQ ID NO:1).

As the natural promoter of nisA is not part of this cassette, theinducible lacA promoter of the lactococcal lactose operon (18, FIG. 5)(SEQ ID NO:3) was employed for expression of nisA and subsequent variantnisA genes. Vector construction involved cloning a XmnI/BamHI fragmentcontaining the lacA promoter, but excluding the lacA ribosome bindingsite, into pTG262 to generate pFI451 (FIG. 4). Into the adjacentpolylinker was cloned the entire nisA cassette on a BamHI/EcoRI fragmentto generate pFI449. The individual components of the nisA gene describedabove ie the 200 bp BamHI/SacI N-fragment and the 254 bp SacI/EcoRIC-fragment, were also separately cloned into pFI451 to generate pFI480and pFI487, respectively (FIG. 4). To recover an intact nisA gene inthese plasmids, insertion of the missing N or C fragment is required.These fragments can be generated by PCR and using appropriate primers,site-specific substitutions can be incorporated into the reconstitutedgene (see below).

Using this expression system the amino acid initially selected foralteration was Serine⁵. In mature nisin A, Serine⁵ is modified to adehydroalanine residue in ring A (FIG. 9) (SEQ ID NO:19). The aim was toreplace this with an Alanine residue by incorporating the requiredmutation in the N-terminal fragment of the nisA cassette using PCRmethodology. Primer 11 (SEQ ID NO:18), which contains an alanine codon(TGC) in place of the Serine⁵ codon (CGA), was used together with primer9 (SEQ ID NO:16) (FIG. 2) to generate an amino-terminal fragmentcarrying this specific mutation. Primer 11 (SEQ ID NO:18) comprisesprimer 10 (SEQ ID NO:17) sequences with an additional 20 nucleotidesincluding an alanine codon in place of the wild type serine codon (FIG.2). Thus, PCR amplification of nisA using primers 9 and 11 (SEQ ID NOS16 and 18, respectively) generated an N-terminal fragment carrying thespecific substitution and subsequent insertion of this BamHI/SacIfragment into pFI487 resulted in a variant nisA gene containing thedesired site-specific mutation. The plasmid encoding this variant nisAgene (nisA/S5A) was designated pFI493.

The Nis⁻ strain FI7332 was transformed with plasmids which encode thenisA gene under the control of either its own promoter (pFI378, FIG. 3c)or the lacA promoter (pFI449, FIG. 4c). When transformants were testedin plate diffusion assays biological activity was detected at a levelapproximately 50% that of the wild-type parent strain FI5876 (FIG. 6.,Table 2). The difference in the expression vector promoters did notappear to significantly affect the level of biological activity (FIG. 6,Table 2).

Transformants of FI7332 containing derivatives of the expression vectorsencoding variant nisA genes were also tested for biological activity.Strains encoding nisA/H27Q and nisA/S5A exhibited nisin activity atlevels lower than that of FI5876, but comparable to those levelsachieved in the expression systems involving nisA complementation (ieFI7332/pFI378 and FI7332/pFI451; Table 2, FIG. 6).

                  TABLE 2                                                         ______________________________________                                        Expression of nisA and variant nisA genes in different host strains.                                          Nisin                                         Strain    Plasmid     Nisin gene                                                                              activity.sup.a                                ______________________________________                                        FI5876    --          nisA      ++                                            MG1614    --          --        -                                             "         pFI172      nisA      -                                             FI7300    --          --        -                                             "         pFI172      nisA      -                                             FI7304    --          --        -                                             FI7330.sup.b                                                                            pFI172      nisA      +                                             FI7332    --          --        -                                             "         pFI172      nisA      +                                             "         pFI354      --        -                                             "         pFI378      nisA      +                                             "         pFI411      nisA/H27G +                                             "         pFI499.sup.c                                                                              nisA      +                                             "         pFI493.sup.c                                                                              nisA/S5A  +                                             ______________________________________                                         .sup.a nisin activity was measured by the plate diffusion assay. Zones of     inhibition of growth were: ++, 24 mm and +, 18-21 mm (including the 8 mm      bore of the well).                                                            .sup.b FI7330 was generated by transforming FI7304 with pFI172 followed b     a chromosomal deletion (see text). As the host strain is no longer FI7304     a new strain number was allocated.                                            .sup.c plasmids are derivatives of pFI451 (FIG. 4a) in which the nisA         cassette is preceded by the lacA promoter. All other plasmids utilise the     natural nisA promoter.                                                   

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 21                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 448 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: join(88..258 - #, 369..446)                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - AACGGNTCNG ATTAAATTCT GAAGTTTGTT AGATACAATG ATTTCGTTCG AA - #GGAACTAC         60                                                                          #TTT AAC TTG        111 ACTCAAA ATG AGT ACA AAA GAT                           #            Met Ser Thr Ly - #s Asp Phe Asn Leu                              #           5  1                                                              - GAT TTG GTA TCT GTT TCG AAG AAA GAT TCA GG - #T GCA TCA CCA CGC ATT          159                                                                          Asp Leu Val Ser Val Ser Lys Lys Asp Ser Gl - #y Ala Ser Pro Arg Ile           #     20                                                                      - ACA AGT ATT NNN CTA TGT ACA CCC GGT TGT AA - #A ACA GGA GCT CTG ATG          207                                                                          Thr Ser Ile Xaa Leu Cys Thr Pro Gly Cys Ly - #s Thr Gly Ala Leu Met           # 40                                                                          - GGT TGT AAC ATG AAA ACA GCA ACT TGT CAN TG - #T AGT ATT CAC GTA AGC          255                                                                          Gly Cys Asn Met Lys Thr Ala Thr Cys Xaa Cy - #s Ser Ile His Val Ser           #                 55                                                          - AAA TAACCAAATC AAAGGATAGT ATTTTGTTAG TTCAGACATG GATACTATC - #C               308                                                                          Lys                                                                           - TATTTTTATA AGTTATTTAG GGTTGCTAAA TAGCTTATAA AAATAAAGAG AG - #GAAAAAAC        368                                                                          - ATG ATA AAA AGT TCA TTT AAA GCT CAA CCG TT - #T TTA GTA AGA AAT ACA          416                                                                          Met Ile Lys Ser Ser Phe Lys Ala Gln Pro Ph - #e Leu Val Arg Asn Thr           #         70                                                                  #         448 CCA AAC GAT AAA CGG ANT TNT AC - #                              Ile Leu Ser Pro Asn Asp Lys Arg Xaa Xaa                                       #     80                                                                      - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 83 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Ser Thr Lys Asp Phe Asn Leu Asp Leu Va - #l Ser Val Ser Lys Lys         #                 15                                                          - Asp Ser Gly Ala Ser Pro Arg Ile Thr Ser Il - #e Xaa Leu Cys Thr Pro         #             30                                                              - Gly Cys Lys Thr Gly Ala Leu Met Gly Cys As - #n Met Lys Thr Ala Thr         #         45                                                                  - Cys Xaa Cys Ser Ile His Val Ser Lys Met Il - #e Lys Ser Ser Phe Lys         #     60                                                                      - Ala Gln Pro Phe Leu Val Arg Asn Thr Ile Le - #u Ser Pro Asn Asp Lys         # 80                                                                          - Arg Xaa Xaa                                                                 - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 546 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - GAAGACTTTC TTTCATAAAG TAATTTTTTT CCAAAGATAA TTCTCTTTTA AT - #TGTATCAT         60                                                                          - AAAAGATAAT ATTTTCAAGG TAAAACAAAC AATTTCAAAC AAAAACAAAC GT - #TAGATGAT        120                                                                          - GAAATAAGAA CAGAGGATTG ACGTATATTA GCTTAGGTCA GATTTTGTAT AA - #GACGAAAA        180                                                                          - TAAAGTAGGA CCTCTTAATC AGTAAGTTAT AGAAAGTAAA AGACTTTTGT AA - #TACCTGAA        240                                                                          - TAGATATTTC ACGTCCATTT TGTGATGGAT TAAATGAACA AAAATGAACA AT - #AATTTAAC        300                                                                          - GGTGTTATCT ATTTTTTAAA AAAACAAATA AAAAAAAACA AAAAATTAAC AA - #AAATAGTT        360                                                                          - GCGTTTTGTT TGAATGTTTG ATATCATATA AACAAAGAAA TGATGAAAAC GT - #TATCTTGA        420                                                                          - ACATTTTGCA AAATATTTTC TACTTCTACG TAGCATTTCG GATCCGATTA AA - #TTCTGAAG        480                                                                          - TTTGTTAGAT ACAATGATTT CGTTCGAAGG AACTACAAAA TAAATTATAA GG - #AGGCACTC        540                                                                          #          546                                                                - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1313 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 90..1262                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - GGTAGTGTAA AATAAGTTGT GTAAACACAA AAAGGAATAA ATCCGTTATA GT - #AGAGTTGC         60                                                                          - GAAACATTAC TAGAAAGAGA TTTATTCCT ATG ACT CAG TTT ACC - # ACA GAA CTA          113                                                                          #              Met Thr G - #ln Phe Thr Thr Glu Leu                            #             5  1                                                            - CTT AAC TTC CTA GCC CAA AAG CAA GAT ATT GA - #T GAA TTT TTC CGT ACT          161                                                                          Leu Asn Phe Leu Ala Gln Lys Gln Asp Ile As - #p Glu Phe Phe Arg Thr           #     20                                                                      - TCT CTT GAA ACT GCT ATG AAT GAT CTG CTT CA - #A GCA GAG TTA TCA GCC          209                                                                          Ser Leu Glu Thr Ala Met Asn Asp Leu Leu Gl - #n Ala Glu Leu Ser Ala           # 40                                                                          - TTT TTA GGG TAT GAA CCT TAC GAT AAA GTA GG - #C TAT AAT TCT GGG AAT          257                                                                          Phe Leu Gly Tyr Glu Pro Tyr Asp Lys Val Gl - #y Tyr Asn Ser Gly Asn           #                 55                                                          - AGT CGT AAC GGA AGC TAT TCA CGG CAA TTT GA - #A ACC AAA TAT GGG ACT          305                                                                          Ser Arg Asn Gly Ser Tyr Ser Arg Gln Phe Gl - #u Thr Lys Tyr Gly Thr           #             70                                                              - GTT CAG TTG AGC ATT CCT AGA GAT CGT AAT GG - #G AAC TTT AGT CCA GCT          353                                                                          Val Gln Leu Ser Ile Pro Arg Asp Arg Asn Gl - #y Asn Phe Ser Pro Ala           #         85                                                                  - TTG CTT CCC GCT TAT GGA CGT CGA GAT GAC CA - #C TTG GAA GAG ATG GTT          401                                                                          Leu Leu Pro Ala Tyr Gly Arg Arg Asp Asp Hi - #s Leu Glu Glu Met Val           #    100                                                                      - ATC AAA CTC TAT CAA ACC GGT GTA ACG ACT CG - #A GAA ATT AGT GAT ATC          449                                                                          Ile Lys Leu Tyr Gln Thr Gly Val Thr Thr Ar - #g Glu Ile Ser Asp Ile           105                 1 - #10                 1 - #15                 1 -       #20                                                                           - ATC GAG CGA ATG TAT GGT CAT CAC TAT AGT CC - #T GCC ACA ATT TCT AAT          497                                                                          Ile Glu Arg Met Tyr Gly His His Tyr Ser Pr - #o Ala Thr Ile Ser Asn           #               135                                                           - ATC TCA AAA GCA ACT CAG GAG AAT GTC GCT AC - #T TTT CAT GAG CGA AGC          545                                                                          Ile Ser Lys Ala Thr Gln Glu Asn Val Ala Th - #r Phe His Glu Arg Ser           #           150                                                               - TTA GAA GCC AAT TAC TCT GTT TTA TTT CTT GA - #C GGA ACC TAT CTT CCA          593                                                                          Leu Glu Ala Asn Tyr Ser Val Leu Phe Leu As - #p Gly Thr Tyr Leu Pro           #       165                                                                   - TTA AGA CGT GGA ACC GTT AGT AAA GAA TGT AT - #T CAT ATC GCA CTT GGC          641                                                                          Leu Arg Arg Gly Thr Val Ser Lys Glu Cys Il - #e His Ile Ala Leu Gly           #   180                                                                       - ATT ACA CCA GAA GGA CAG AAG GCT GTT CTT GG - #A TAT GAA ATC GCC CCA          689                                                                          Ile Thr Pro Glu Gly Gln Lys Ala Val Leu Gl - #y Tyr Glu Ile Ala Pro           185                 1 - #90                 1 - #95                 2 -       #00                                                                           - AAT CAA AAT AAT GCT TCT TGG TCC ACC CTG TT - #A GAC AAG CTT CAA AAC          737                                                                          Asn Gln Asn Asn Ala Ser Trp Ser Thr Leu Le - #u Asp Lys Leu Gln Asn           #               215                                                           - CAA GGA ATC CAA CAG GTT TCT CTT GTA GTG AC - #C GAT GGC TTC AAG GGG          785                                                                          Gln Gly Ile Gln Gln Val Ser Leu Val Val Th - #r Asp Gly Phe Lys Gly           #           230                                                               - CTT GAA CAG ATT ATC AGT CAG GCT TAC CCA TT - #A GCT AAA CAA CAA CGT          833                                                                          Leu Glu Gln Ile Ile Ser Gln Ala Tyr Pro Le - #u Ala Lys Gln Gln Arg           #       245                                                                   - TGC TTA ATT CAT ATT AGT CGA AAT CTA GCT AG - #T AAA GTG AAA CGA GCA          881                                                                          Cys Leu Ile His Ile Ser Arg Asn Leu Ala Se - #r Lys Val Lys Arg Ala           #   260                                                                       - GAT AGA GCG GTT ATT CTG GAG CAA TTT AAA AC - #G ATT TAT CGT GCT GAA          929                                                                          Asp Arg Ala Val Ile Leu Glu Gln Phe Lys Th - #r Ile Tyr Arg Ala Glu           265                 2 - #70                 2 - #75                 2 -       #80                                                                           - AAT TTA GAA ATG GCA GTG CAA GCT TTA GAG AA - #C TTT ATC GCC GAA TGG          977                                                                          Asn Leu Glu Met Ala Val Gln Ala Leu Glu As - #n Phe Ile Ala Glu Trp           #               295                                                           - AAA CCA AAG TAT AGG AAA GTC ATG GAA AGT CT - #G GAG AAT ACG GAT AAT         1025                                                                          Lys Pro Lys Tyr Arg Lys Val Met Glu Ser Le - #u Glu Asn Thr Asp Asn           #           310                                                               - CTT TTA ACT TTT TAT CAG TTT CCC TAC CAG AT - #T TGG CAC AGC ATT TAT         1073                                                                          Leu Leu Thr Phe Tyr Gln Phe Pro Tyr Gln Il - #e Trp His Ser Ile Tyr           #       325                                                                   - TCG ACA AAC CTC ATT GAG TCT CTT AAC AAA GA - #A ATC AAA CGT CAA ACG         1121                                                                          Ser Thr Asn Leu Ile Glu Ser Leu Asn Lys Gl - #u Ile Lys Arg Gln Thr           #   340                                                                       - AAA AAG AAG GTT CTT TTT CCT AAC GAG GAG GC - #T CTG GAA CGT TAC TTA         1169                                                                          Lys Lys Lys Val Leu Phe Pro Asn Glu Glu Al - #a Leu Glu Arg Tyr Leu           345                 3 - #50                 3 - #55                 3 -       #60                                                                           - GTT ACT TTG TTT GAA GAT TAT AAT TTC AAG CA - #A AGT CAA CGC ATC CAT         1217                                                                          Val Thr Leu Phe Glu Asp Tyr Asn Phe Lys Gl - #n Ser Gln Arg Ile His           #               375                                                           - AAA GGG TTT GGC CAA TGT GCT GAC ACA CTT GA - #A AGC TTA TTT GAT             1262                                                                          Lys Gly Phe Gly Gln Cys Ala Asp Thr Leu Gl - #u Ser Leu Phe Asp               #           390                                                               #           1313CTCTACT TGAGTGTTTA CACATAATTA TTGACAGTAT C                    - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 391 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Met Thr Gln Phe Thr Thr Glu Leu Leu Asn Ph - #e Leu Ala Gln Lys Gln         #                 15                                                          - Asp Ile Asp Glu Phe Phe Arg Thr Ser Leu Gl - #u Thr Ala Met Asn Asp         #             30                                                              - Leu Leu Gln Ala Glu Leu Ser Ala Phe Leu Gl - #y Tyr Glu Pro Tyr Asp         #         45                                                                  - Lys Val Gly Tyr Asn Ser Gly Asn Ser Arg As - #n Gly Ser Tyr Ser Arg         #     60                                                                      - Gln Phe Glu Thr Lys Tyr Gly Thr Val Gln Le - #u Ser Ile Pro Arg Asp         # 80                                                                          - Arg Asn Gly Asn Phe Ser Pro Ala Leu Leu Pr - #o Ala Tyr Gly Arg Arg         #                 95                                                          - Asp Asp His Leu Glu Glu Met Val Ile Lys Le - #u Tyr Gln Thr Gly Val         #           110                                                               - Thr Thr Arg Glu Ile Ser Asp Ile Ile Glu Ar - #g Met Tyr Gly His His         #       125                                                                   - Tyr Ser Pro Ala Thr Ile Ser Asn Ile Ser Ly - #s Ala Thr Gln Glu Asn         #   140                                                                       - Val Ala Thr Phe His Glu Arg Ser Leu Glu Al - #a Asn Tyr Ser Val Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Phe Leu Asp Gly Thr Tyr Leu Pro Leu Arg Ar - #g Gly Thr Val Ser Lys         #               175                                                           - Glu Cys Ile His Ile Ala Leu Gly Ile Thr Pr - #o Glu Gly Gln Lys Ala         #           190                                                               - Val Leu Gly Tyr Glu Ile Ala Pro Asn Gln As - #n Asn Ala Ser Trp Ser         #       205                                                                   - Thr Leu Leu Asp Lys Leu Gln Asn Gln Gly Il - #e Gln Gln Val Ser Leu         #   220                                                                       - Val Val Thr Asp Gly Phe Lys Gly Leu Glu Gl - #n Ile Ile Ser Gln Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Tyr Pro Leu Ala Lys Gln Gln Arg Cys Leu Il - #e His Ile Ser Arg Asn         #               255                                                           - Leu Ala Ser Lys Val Lys Arg Ala Asp Arg Al - #a Val Ile Leu Glu Gln         #           270                                                               - Phe Lys Thr Ile Tyr Arg Ala Glu Asn Leu Gl - #u Met Ala Val Gln Ala         #       285                                                                   - Leu Glu Asn Phe Ile Ala Glu Trp Lys Pro Ly - #s Tyr Arg Lys Val Met         #   300                                                                       - Glu Ser Leu Glu Asn Thr Asp Asn Leu Leu Th - #r Phe Tyr Gln Phe Pro         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Tyr Gln Ile Trp His Ser Ile Tyr Ser Thr As - #n Leu Ile Glu Ser Leu         #               335                                                           - Asn Lys Glu Ile Lys Arg Gln Thr Lys Lys Ly - #s Val Leu Phe Pro Asn         #           350                                                               - Glu Glu Ala Leu Glu Arg Tyr Leu Val Thr Le - #u Phe Glu Asp Tyr Asn         #       365                                                                   - Phe Lys Gln Ser Gln Arg Ile His Lys Gly Ph - #e Gly Gln Cys Ala Asp         #   380                                                                       - Thr Leu Glu Ser Leu Phe Asp                                                 385                 3 - #90                                                   - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 100 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "promoter" DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - ACGTGCTATC ACTTGAAGCT TATTGATTAA TATTCTTCAA CTCTACTTGA GT - #GTTTACAC         60                                                                          #   100            TCCA ATTCTTATCT CTTTTCAATA                                 - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 100 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "promoter" DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - ACGTGCTATC ACTTGAAGCT TATTGATTAA TATTCTTCAA CTCTACTGCA AC - #TTGTCATT         60                                                                          #   100            CAAA TAACCAAATC AAAGGATAGT                                 - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #   17             T                                                          - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 16 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                # 20               ATTG                                                       - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #  18              TG                                                         - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #               25 CGTT TGGAG                                                 - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #                24GTAT TCAC                                                  - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #                24CAAG TTGC                                                  - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #             28   TTCT GAAGTTTG                                              - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                # 20               ACAA                                                       - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #    40            ACAA CCGGGTGTAC ATAGTGCAAT                                 - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 34 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/product= "Dhb"HER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/product= "Dha or Ala"ORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/product= "Abu"HER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/product= "Abu"HER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 23                                                    #/product= "Abu"HER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 25                                                    #/product= "Abu"HER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 27                                                    #/product= "His or Gln"ORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 33                                                    #/product= "Dha"HER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Ile Xaa Ala Ile Xaa Leu Ala Xaa Pro Gly Al - #a Lys Xaa Gly Ala Leu         #                15                                                           - Met Gly Ala Asn Met Lys Xaa Ala Xaa Ala Xa - #a Ala Ser Ile His Val         #            30                                                               - Xaa Lys                                                                     - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 100 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "promoter" DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - TATTGAAAAG AGATAAGAAT TGGATACTGT CAATAATTAT GTGTAAACAC TC - #AAGTAGAG         60                                                                          #   100            AATA AGCTTCAAGT GATAGCACGT                                 - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 100 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "promoter" DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - ACTATCCTTT GATTTGGTTA TTTCGTTACG TGAATGCTAC AATGACAAGT TG - #CAGTAGAG         60                                                                          #   100            AATA AGCTTCAAGT GATAGCACGT                                 __________________________________________________________________________

We claim:
 1. An organism which does not secrete a natural nisA nisin,but which expresses genes for nisin modification, immunity, andtranslocation out of the cell, wherein the organism is a lactococcalstrain.
 2. The organism according to claim 1 transformed with a codingsequence for a variant prenisin and, if necessary, appropriateregulatory sequences for expression thereof such that the organismsecretes the variant nisin corresponding to the variant prenisin.
 3. Amethod of producing the organism of claim 1 comprising selecting a nisinproducing organism which contains a coding sequence for its natural nisAgene product and selectively deleting the coding sequence for itsnatural nisA gene product.
 4. A method of producing the organism ofclaim 1 comprising selecting a nisin producing organism which contains anisA gene and insertionally inactivating the nisA gene and restoring theactivity of the genes for nisin modification, immunity, andtranslocation out of the cell.
 5. The method according to claim 4wherein the restoration of the activity of the genes for nisinmodification, immunity, and translocation out of the cell is achieved byselection in media containing nisin.
 6. A method according to any one ofclaims 3, 4 and 5 further comprising transforming the organism with acoding sequence for a variant prenisin and, if necessary, appropriateregulatory sequences for expression thereof, such that the organismsecretes the variant nisin corresponding to the variant prenisin.
 7. Aprocess for producing a nisin comprising fermenting the organism ofclaim 2 and obtaining the nisin produced thereby.
 8. The processaccording to claim 7 wherein the lactococcal strain is Lactococcuslactis.
 9. The process according to claim 7 wherein the coding sequencefor said variant prenisin comprises a codon other than a serine codoncorresponding to a codon for a serine residue in unmodified prenisinwhich is converted to dehydroalanine in a first ring of mature nisin.10. The process according to claim 8 wherein the coding sequence forsaid variant prenisin comprises a codon other than a serine codoncorresponding to a codon for a serine residue in unmodified prenisinwhich is converted to dehydroalanine in a first ring of mature nisin.11. The process according to claim 9 wherein said codon other than aserine codon is an alanine codon.
 12. The process according to claim 10wherein said codon other than a serine codon is an alanine codon.
 13. Anorganism which is a Lactococcus lactis which does not secrete itsnatural nisA nisin, but which expresses genes for nisin modification,immunity, and translocation out of the cell.
 14. The organism accordingto claim 13 transformed with a coding sequence for a variant prenisinand, if necessary, appropriate regulatory sequences for expressionthereof such that the organism secretes the variant nisin correspondingto the variant prenisin.
 15. A method of producing the organism of claim13 comprising selecting a nisin producing organism which contains acoding sequence for its natural nisA gene product and selectivelydeleting the coding sequence for its natural nisA gene product.
 16. Amethod of producing the organism of claim 13 comprising selecting anisin producing organism which contains a nisA gene and insertionallyinactivating the nisA gene and restoring the activity of the genes fornisin modification, immunity and translocation out of the cell.
 17. Themethod according to claim 16 wherein the restoration of the activity ofthe genes for nisin modification, immunity and translocation out of thecell is achieved by selection in media containing nisin.
 18. The methodaccording to any one of claims 15, 16 and 17 further comprisingtransforming the organism with a coding sequence for a variant prenisinand, if necessary, appropriate regulatory sequences for expressionthereof, such that the organism secretes the variant nisin correspondingto the variant prenisin.